Antibodies constitute a growing class of drugs that are being administered for treatment of an increasing range of human diseases, including but not limited to autoimmunity, infection and cancer. While engineering antibodies to recognize virtually any antigen has become technologically straightforward, engineering antibodies to induce distinct immune signals, or effector functions, which direct the killing of cells in vivo remains technologically challenging. This latter property is carried out by the Fc region of antibodies and the difficulty in engineering antibody Fc regions is due to the presence of a conserved N-linked glycan attached to Asn297 in clinically- relevant IgG antibodies. The next generation of immunotherapeutic antibodies, as well as our abilities to identify and understand antibody-mediated killing mechanisms, depends on our ability to rationally modify the chemical structure of this Asn297-linked glycan. The most important molecular feature of this glycan is a fucose sugar unit connected through an ?(1,6) linkage to the Asn-proximal N-actylglucosamine (GlcNac), the absence of which imparts Fc domains with increased binding affinity to an activating Fc?R, Fc?R3A, resulting in substantially increased antibody-mediated in vivo cellular killing. Based on our preliminary studies of AlfC, an ?(1,6)- fucosidase that removes fucose from Asn297-linked glycans on IgG antibodies, but only after all of the branched sugar units beyond the GlcNac to which it is linked have been removed, we propose to develop ?(1,6)-fucosidase variants that can rapidly, reliably and entirely remove the fucose sugar unit on any antibody, regardless of the branched structure of the Asn297-linked glycan. Such an enzymatic tool could be used by the immunological community to evaluate the in vivo antibody-mediated killing mechanisms of the entire catalog of antibodies, both currently available and to be developed in the future. In this proposal, we will address two Specific Aims: (i) to define the molecular basis of antibody defucosylation by ?(1-6)-fucosidases; and (ii) to design ?(1,6)-fucosidase variants active on antibodies bearing fully branched glycans. Progress towards these complementary, yet independent, Specific Aims will significantly advance our understanding of glycan-modifying enzymes. Leveraging this knowledge in the context of AlfC and related ?(1-6)-fucosidases will enhance our ability to customize antibodies, providing the tools with which immunologists can better understand antibody-mediated in vivo cellular killing, as well as further unleashing their vast therapeutic utility and expanding their positive impact on human health.